QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (34) Citing Articles via Google Scholar Google Scholar Articles by Jacobs, J. M. Articles by Meyer, T. Search for Related Content PubMed PubMed Citation Articles by Jacobs, J. M. Articles by Meyer, T.

 Previous Article  |  Next Article 

Volume 17, Number 11, Issue of June 1, 1997 pp. 4129-4135
Copyright ©1997 Society for Neuroscience

Control of Action Potential-Induced Ca²⁺ Signaling in the Soma of Hippocampal Neurons by Ca²⁺ Release from Intracellular Stores

Jason M. Jacobs and Tobias Meyer

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

REFERENCES

---

ABSTRACT

Stimulus-induced increases in neuronal Ca²⁺ concentration are important signaling events for transcriptional regulation and neuronal plasticity. Electrical inputs are thought to mediate Ca²⁺ responses in the soma by triggering action potentials, which in turn open voltage-gated Ca²⁺ channels in the somatic plasma membrane. It is not yet known to what extent internal Ca²⁺ amplification contributes to the somatic Ca²⁺ responses. Here we used fluorescent Ca²⁺ measurements in cultured hippocampal neurons and report that the amplitude of the somatic Ca²⁺ increase triggered by field stimulation is independent of the extracellular Ca²⁺ concentration as long as the concentration is greater than 50 µM. Furthermore, significantly more La³⁺ has to be added extracellularly for blocking Ca²⁺ responses, as predicted from the reported La³⁺ dependence of voltage-gated Ca²⁺ channels. These measurements suggest that field stimulation-induced somatic Ca²⁺ responses in hippocampal neurons are largely attributable to Ca²⁺ release from intracellular stores. Only a small number of Ca²⁺ ions have to enter across the plasma membrane for this intracellular Ca²⁺ amplification process to occur. Rapid fluorescence-imaging measurements showed that the internal Ca²⁺ amplification occurs over 10-15 msec and linearly increases intracellular Ca²⁺ concentrations for up to 40 action potentials. At a fixed number of field pulses, frequencies of 40 Hz were optimal for somatic Ca²⁺ increases. Our studies suggest that the opening of intracellular Ca²⁺ release channels plays a crucial part in shaping the action potential-induced neuronal Ca²⁺ response.

Key words: hippocampal neurons; action potential; calcium release; field stimulation; Fluo3; calcium amplification

---

INTRODUCTION

Ca²⁺ signaling in neurons is important for neurotransmitter release, synaptic plasticity (Malenka, 1994; Malgaroli, 1994), and transcriptional control (Frank and Greenberg, 1994; Deisseroth et al., 1996). Although presynaptic and postsynaptic Ca²⁺ signals have been studied extensively (e.g., Augustine and Neher, 1992; Neher, 1993), less is known about the role and mechanism of action potential-induced Ca²⁺ increases in the soma. A likely function of such Ca²⁺ responses is to transduce information about the electrical activity into processes within the nucleus, because depolarization-mediated somatic Ca²⁺ increases have been shown to alter gene expression as well as other somatic processes (Frank and Greenberg, 1994; Deisseroth et al., 1996). In one Ca²⁺-dependent signal transduction pathway, transcriptional activation can be mediated by Ca²⁺/calmodulin-dependent protein kinase II- or IV-mediated phosphorylation of cAMP response element binding protein. The question arises of how the short-lasting Ca²⁺ influx during an action potential can sufficiently increase cytosolic and nuclear Ca²⁺ concentrations to activate the transcriptional machinery.

Here we investigate the significance of internal Ca²⁺ release for action potential-induced somatic Ca²⁺ transients. Similar to the internal Ca²⁺ amplification mechanisms in skeletal and cardiac muscle, an action potential-induced intracellular Ca²⁺ release process may lead to a much larger amplitude of the somatic Ca²⁺ response than could be attained by Ca²⁺ influx alone. In addition to a role in increasing the amplitude of Ca²⁺ transients, action potential-induced opening of intracellular Ca²⁺ release channels would also likely be important in mediating selective activation of enzymes and transcription factors that are colocalized with such release channels.

---

MATERIALS AND METHODS

Cell culturing. Primary hippocampal cultures were prepared using a modification of the procedure described by Ryan and Smith (1995). Hippocampal regions were removed from day 3 rat pups in Ca²⁺- and Mg²⁺-free HBSS, and the CA1-CA3 region was subdissected from the dentate gyrus and subicular structures. The CA1-CA3 region was then collected, minced, and incubated for 10 min at 37°C in the presence of 0.25% trypsin, 1 mg/ml DNase (both from Sigma, St. Louis, MO), and 10 mM Mg²⁺. The cells were then triturated and plated onto acid-washed coverslips that had been coated previously with 0.01% poly-D-lysine (Sigma) and 1:100 Matrigel (Collaborative Research, Bedford, MA). Media consisted of minimum essential medium supplemented with 2% B27, 6% fetal calf serum (all from Life Technologies, Gaithersburg, MD), 30 mg/L insulin, and 100 mg/L transferrin (Sigma). Cell densities at plating were 30,000-50,000 cells/cm². At 48 hr in culture cytosine b-D-arabinofuranoside (Sigma) was added to a final concentration of 6 µM. Feeding consisted of exchanging 50% of the media twice a week. Cultures were incubated in a 37°C humidified chamber maintained at 5% CO₂. For all experiments, the neurons were used 2-5 weeks after plating. It has been shown that functional synapses exist in primary hippocampal cultures of this age (Basarsky et al., 1994). A chamber was formed by attaching to the coverslip a Teflon ring (inner diameter, 1.2 cm; height, 2-3 mm; total volume, ~300 µl), using silicone vacuum grease as a sealant. During experiments the culturing medium was switched to a standard saline solution consisting of 135 mM NaCl, 5 mM KCl, 1.5 mM CaCl₂, 1.5 mM MgCl₂, 20 mM HEPES (buffered to pH 7.4), 10 mM glucose, 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Research Biochemicals International, Natick, MA), and 250 µM sulfinpyrazone (Sigma). The dye-loading solution consisted of 5 µM Fluo3-AM (Molecular Probes, Eugene, OR) in the standard saline, with the omission of CNQX. The neurons were allowed to remain in the dye-loading solution for 20 min before being washed in the standard saline and used for experiments. Neurons with triangular or fusiform morphology, which are mostly either CA1 or CA3 type neurons (and not interneurons), were analyzed in these studies. All experiments were performed at room temperature (~25°C).

Field stimulation. Ca²⁺ transients were stimulated with a standard protocol by lowering two parallel platinum electrodes (3 mm apart) into the chamber in near contact with the surface and then passing 1 msec current pulses between the electrodes, yielding fields of ~10 V/cm across the surface of the coverslip. The standard protocol was a 1-sec-long train, or "burst," of 30 current pulses (i.e., 30 Hz). A 486 IBM-compatible computer with analog-to-digital and digital-to-analog conversion boards was used along with a high voltage supply and self-built electronics to allow computer control of the timing and strength of the applied electric field pulses as well as for monitoring the amount of current applied to the cells. From the measured current, the electric field strength was calculated via a mathematical model using the known resistivity of saline:  = 60 ·cm. Each measurement in Figure 1, C and D, involved six bursts. Three identical bursts were triggered 15 sec apart and were followed by three reference bursts (reference conditions: 10 V/cm; pulse width, 1 msec; 30 pulses over 1 sec). A relative peak amplitude of the experimental fluorescence was calculated by dividing the average amplitude of the fluorescence peak of the initial three responses by the averaged reference amplitude. All errors are plotted as SEM. Best fits were calculated via the Levenberg-Marquardt method using Deltagraph software (Deltapoint, Monterey, CA).

---

Fig. 1. Somatic Ca²⁺ responses triggered by field stimulation. A, Transient increases in intracellular [Ca²⁺] are triggered by six trains of 30 field pulses applied at 30 Hz. Tracings from three cells are shown. Ca²⁺ responses were recorded in the soma of primary cultured hippocampal neurons loaded with the fluorescent Ca²⁺ indicator Fluo3. The relative increase in fluorescence of each response was ~1.5. B, The amplitude of the Ca²⁺ response increased with the number of field pulses applied per train. Pulses were also applied at 30 Hz. C, The peak amplitude of the relative fluorescence versus the applied electric field strength is shown for an individual cell. A sharp threshold for triggering Ca²⁺ responses can be observed. Maximal responses typically required 10 V/cm. The line is the best fit to the data of the equation: A = aE_1/2ⁿ/(E_1/2ⁿ + E_1/2ⁿ), where A as the relative amplitude, a is the maximal amplitude, E_1/2 = 6.0 V/cm, and n = 40. D, [Ca²⁺] responses also show an all-or-none dependence on the duration of the field pulse. Pulses longer than 0.2 msec were typically required for triggering a [Ca²⁺] rise. No further [Ca²⁺] increase was observed by increasing the duration past 0.2 msec. The line is the best fit to the data with the form: A = aⁿ/(ⁿ + _1/2ⁿ), where _1/2 = 0.13 msec, and n = 10.4.
[View Larger Version of this Image (25K GIF file)]

---

Fluorescence imaging. The Ca²⁺ indicator Fluo3-AM was excited at 488 nm using an inverted Nikon Diaphot microscope and an Odyssey confocal imaging system with a 100 µm confocal slit (Noran Instruments, Middleton, WI). Fluorescence intensity traces from individual neurons were obtained by monitoring the average overall intensity of the soma of each cell every 100 msec by using Image-1 software (Universal Imaging Corporation, West Chester, PA). In the experiments depicted in Figure 3, a Raptor imaging board (Bitflow, Woburn, MA) and Eye Image Calculator software (IO Industries, London, Ontario, Canada) were used to capture delaced images from the confocal microscope every 16.7 msec. A second laser coupled into the optical axis was used to create a 2 msec timing pulse. This timing pulse was used to determine the onset of the field pulse and to confirm the line scan time. In this experiment, 1-msec-long field pulses were applied every 200 msec. A baseline fluorescence image was determined by averaging the 10 images before the onset of stimulation. This baseline was subtracted from all subsequent images. The images were scaled to a uniform signal height, and the average brightness in the vicinity of the plasma membrane was determined line by line. The analysis was completed by inserting the 2.3 msec interval between frames into the data, followed by rescaling to adjust for baseline and signal height. Only those traces with signal-to-noise ratios better than 50% of that of the best trace were used.

---

Fig. 3. Spatial and temporal analysis of action potential-induced Ca²⁺ responses. Intracellular Ca²⁺ release occurs over 10-15 msec and originates mostly from Ca²⁺ stores close to the plasma membrane. A, Rise in fluorescence intensity induced by a single action potential in the soma of an individual neuron as determined by a confocal microscope in line scan analysis mode with a 60 µsec scan time (green, low fluorescence; red, high fluorescence). The trace begins ~2 msec after the onset of the applied electric field pulse. The greatest rise in [Ca²⁺]_i occurs near the plasma membrane. B, Rise in fluorescence intensity near the plasma membrane for a single action potential in three separate neurons. The time scale represents milliseconds after the onset of the applied electric field pulse, and the shaded box represents the duration of the field pulse. The small overshoot in fluorescence at a t of approximately 3 msec resulted from error from a photomultiplier tube after an ultraviolet timing pulse. The interruptions seen resulted from 2.3 msec pauses in the data collection every 14.3 msec. The solid line is the best fit to the data of an equation of the form: F(t) = atⁿ/(t_1/2ⁿ + tⁿ), where t_1/2 = 4.9 msec , and n = 3.3. The individual fluorescence traces were statistically indistinguishable from the average (for individual traces, t_1/2 = 4.6 ± 0.3, and n = 3.7 ± 0.3). C, Average fluorescence rise induced in the soma near the plasma membrane by a single action potential (n = 16 action potentials in nine distinct neurons). The shaded box again represents the duration of the electric field pulse, and the small overshoot at a t of approximately 3 msec represents photomultiplier tube error. The solid line is the best fit to the data of an equation of the form: F(t) = atⁿ/(t_1/2ⁿ + tⁿ). The dashed line is the first derivative of the calculated curve fit and demonstrates that the highest rate of rise occurs 3-5 msec after the onset of the field pulse, and a full Ca²⁺ rise requires 10-15 msec.
[View Larger Version of this Image (47K GIF file)]

---

Perfusion of cultured neurons. Flow exchange in the chamber was accomplished with a gravity feed system of three solutions connected to a common output via a series of computer-controlled switches. The output was allowed to flow into one side of the chamber, and a suction device was placed above the surface of the saline solution on the opposite side of the chamber to drain fluid during flow. Computer-controlled mixing of solutions allowed a variety of Ca²⁺ concentrations to be achieved. The flow rate was ~1.4 ml/min, and the chamber held ~300 µl. The standard time for fluid exchange used was as long as 70-80 sec to prevent mechanical perturbance. The chamber contents could be exchanged to >98% in 70 sec, as verified by measuring the fluorescence of a 10 µM fluorescein solution. A standard saline solution with a [Ca²⁺] of 300 nM was made by adding 1 mM EGTA to standard saline with no added Ca²⁺, and the [Ca²⁺] was verified by using the fluorescent indicator indomethacin 1 in a fluorescence spectrophotometer. Similarly, a standard saline solution with a [Ca²⁺] of 30 nM was made by adding 10 mM EGTA. Each measurement in Figure 2 consisted of six bursts. Two reference bursts at a [Ca²⁺]_e of 1.5 mM were followed by a full fluid exchange to a desired [Ca²⁺]_e, two more bursts, a full fluid exchange back to a [Ca²⁺]_e of 1.5 mM, and two final bursts. Burst parameters were the same as in Figure 1. A relative peak amplitude of fluorescence was calculated by dividing the average amplitude of the fluorescence peaks of the experimental condition by the linear interpolation of the relative peak amplitudes of the reference conditions. This procedure helped to correct for dye bleaching over these relatively long experiments. Each measurement in Figure 2C also consisted of six bursts. Two reference bursts in standard saline with no lanthanum present were followed by a full fluid exchange to standard saline with the desired [La³⁺] and then two experimental bursts. The experiment was completed with a 90% fluid exchange with standard saline without added Ca²⁺ and with 1 mM EGTA followed immediately by a full fluid exchange back to standard saline and two final reference bursts.

---

Fig. 2. Dependence of somatic Ca²⁺ responses on extracellular Ca²⁺ concentration. A, The action potential-induced intracellular Ca²⁺ rise is independent of external Ca²⁺ concentration from 500 to 3000 µM (data points and solid line). The dashed line illustrates the expected results of this experiment under the assumption that the intracellular Ca²⁺ rise was the direct result of Ca²⁺ influx from voltage-gated Ca²⁺ channels. B, Only when external [Ca²⁺] is lowered below 50 µM does the action potential-induced Ca²⁺ rise become diminished. C, Dependence of somatic Ca²⁺ responses on extracellular lanthanum concentration. La³⁺ suppressed the action potential-induced intracellular Ca²⁺ rise but with a much higher IC₅₀ (13 µM) than expected. An extracellular [Ca²⁺] of 1.5 mM was present throughout. The dashed line again represents the expectation based on the assumption of the Ca²⁺ rise coming directly from influx via voltage-gated Ca²⁺ channels, modeled on data from the effect of La³⁺ on individual voltage-gated Ca²⁺ channels, with an IC₅₀ of 1.7 µM. The solid line is the best fit to the data with the form: A = aIC₅₀/([La³⁺] + IC₅₀), where IC₅₀ = 13 µM, and a = 262 nM.
[View Larger Version of this Image (17K GIF file)]

---

Calibration of Ca²⁺ concentration. The relative peak amplitude of fluorescence was converted to an apparent free intracellular [Ca²⁺] via a calibration performed on a similar neuron culture (n = 10 neurons), and repeated on several occasions with similar results. This calibration was accomplished via the equation [Ca²⁺] = K_d [F(t)  F_min)/(F_max  F(t)], where F(t) is the measured fluorescence intensity of Fluo3, K_d = 316 nM (Molecular Probes), and F_max was measured as the fluorescence intensity after addition of ionomycin (~1 µM) in the presence of an extracellular [Ca²⁺] of 1.5 mM. F_min was calculated as 0.02% of F_max. Background autofluorescence was negligible compared with the fluorescence of Fluo3-AM-loaded neurons. The background dark current was measured with the excitation laser blocked and was subtracted from F, F_min, and F_max.

---

RESULTS

Action potentials and Ca²⁺ responses induced by field stimulation

We investigated Ca²⁺ signals in the soma of primary cultured hippocampal neurons by field stimulation. Field stimulation of these cells has been used before, but no description of the relevant parameter ranges for field stimulation has been determined (Ryan and Smith, 1995; Deisseroth et al., 1996). First, we measured Ca²⁺ increases in response to field stimuli of different amplitudes and durations. Hippocampal neurons loaded with the fluorescent Ca²⁺ indicator Fluo3-AM responded with a significant increase in free [Ca²⁺]_i in the soma to each train of 30 electric field pulses applied at 30 Hz (Fig. 1A, tracings from three cells shown). Each Ca²⁺ transient reflects the net effect of 30 field pulses. The amplitude of the Ca²⁺ response increased with the number of field pulses applied per train (Fig. 1B, one cell shown). The first Ca²⁺ transient in Figure 1B was generated by two field pulses, and the last transient was generated by 16 field pulses. The increase in [Ca²⁺]_i showed an all-or-none type dependence on the applied electric field strength (Fig. 1C, typical result from an individual neuron). The voltage dependence was sharper for individual neurons than it was for the average of an ensemble of neurons, probably because of differences in the local electric field that each neuron experienced. This could be expressed best by fitting an apparent cooperativity coefficient to the data: n = 40 for the graph of the individual neuron shown (n = 26 ± 12 on average for five individual neurons). The same kind of all-or-none dependence was seen with variations of the pulse width (Fig. 1D). The high apparent cooperativity in the two analyses strongly suggests that the observed Ca²⁺ responses are indeed the result of field stimulation-induced action potentials. Most likely, each 1 msec field pulse induces a single action potential in this preparation.

In an additional control, application of 1 µM tetrodotoxin, an inhibitor of voltage-sensitive Na⁺ channels, reversibly suppressed these [Ca²⁺]_i increases (data not shown). This all-or-none dependence and the requirement for functioning Na⁺ channels suggests that an applied short electric field pulse induces an individual action potential, which in turn leads to a [Ca²⁺]_i increase.

Extracellular Ca²⁺ dependence of Ca²⁺ responses

It is of functional importance whether the action potential-induced [Ca²⁺]_i rise in the soma results from Ca²⁺ influx or from internal release of Ca²⁺, because internal release may increase the amplitude of Ca²⁺ signals significantly. We determined the significance of internal stores for Ca²⁺ release by varying the extracellular Ca²⁺ concentration. Studies have shown that the Ca²⁺ flux conducted by voltage-gated Ca²⁺ channels has a linear dependence on the extracellular Ca²⁺ concentration and shows half-maximal saturation between 14 and 50 mM, depending on the cell type and experimental method used (Frankenhaeuser and Hodgkin, 1957; Okamoto et al., 1976; Kawa, 1979; Hagiwara and Byerly, 1981). Therefore, if somatic Ca²⁺ rises result directly from Ca²⁺ influx, a linear intracellular Ca²⁺ response would be expected below an extracellular Ca²⁺ concentration of 10 mM.

Surprisingly, no change in the amplitude of the Ca²⁺ response was observed when the extracellular Ca²⁺ concentration was varied between 0.5 and 3.0 mM (Fig. 2A). The dashed line represents expected results based on the assumption of the Ca²⁺ rise coming directly from influx via voltage-gated Ca²⁺ channels (assuming linear dependence on the external calcium concentration). The contrasting solid line is the best fit to the data. Even when the extracellular Ca²⁺ concentration was lowered to 50 µM or increased to 10 mM, the amplitude of the response was not changed (Fig. 2B). Only below 50 µM, more than 30-fold below normal, was there a decrease in the action potential-induced Ca²⁺ response. Again, the dashed line in Fig. 2B represents expected results based on the assumption of the Ca²⁺ rise coming directly from influx via voltage-gated Ca²⁺ channels. These data are inconsistent with direct influx of Ca²⁺ as the main mechanism for the action potential-induced Ca²⁺ rise. Although earlier reports suggested that the time course of action potentials can be affected by large changes in extracellular Ca²⁺ concentration (Frankenhaeuser and Hodkin, 1957), it is unlikely that alterations in the properties of action potentials can explain this Ca²⁺ independence over such a large range of extracellular Ca²⁺ concentrations.

Lanthanum dependence of Ca²⁺ responses

To investigate further whether the rise in [Ca²⁺]_i is a direct result of Ca²⁺ influx via voltage-gated Ca²⁺ channels, we tested whether blocking voltage-gated Ca²⁺ channels with La³⁺ affects the action potential-induced Ca²⁺ rise. Earlier studies have shown that La³⁺ is a potent blocker of voltage-gated Ca²⁺ channels, with IC₅₀ values of 1.7 µM for transient currents and 0.14 µM for sustained currents (Boland et al., 1991). Figure 2C shows that 13 µM [La³⁺] is required for half-maximal suppression of Ca²⁺ responses. Thus, [La³⁺] has to be increased nearly 8- to 100-fold above the IC₅₀ for blocking of individual voltage-gated Ca²⁺ channels for inhibition of the intracellular Ca²⁺ response. The dashed line in Figure 2C represents the expectation based on the assumption of the Ca²⁺ rise coming directly from influx via voltage-gated Ca²⁺ channels (using the IC₅₀ reported previously for transient currents).

Interpretation of Figure 2C yields additional information regarding the mechanism of the Ca²⁺ rise. Between 90 and 99% of the individual voltage-gated Ca²⁺ channels are blocked at an [La³⁺] of 13 µM (Boland et al., 1991), but the action potential-induced Ca²⁺ rise was only reduced by 50%. This disparity implies that direct influx of Ca²⁺ via voltage-gated Ca²⁺ channels cannot be the main mechanism responsible for the Ca²⁺ rise. Together, the observations of Figure 2 strongly suggest that, although some Ca²⁺ influx is necessary, Ca²⁺ influx across the plasma membrane alone is not sufficient for a significant somatic Ca²⁺ rise to occur. In other words, each action potential causes voltage-gated Ca²⁺ channels to open, and the resulting Ca²⁺ influx triggers a much larger Ca²⁺ rise from other sources. The observed reduction of the maximal Ca²⁺ response for extracellular Ca²⁺ concentrations below 50 µM suggests that the Ca²⁺ influx through voltage-gated Ca²⁺ channels leads to a more than 30-fold amplification of the Ca²⁺ response by opening Ca²⁺ release channels in intracellular Ca²⁺ stores.

Because ryanodine receptors have been identified in hippocampal neurons by immunohistochemistry (Seymour-Laurent and Barish, 1995), we tested their potential involvement in Ca²⁺ release by applying 20 mM caffeine for up to 25 min and 10 µM ryanodine for up to 20 min to the neurons. At least for these concentrations, no apparent change in the amplitude of action potential-induced Ca²⁺ responses could be observed (data not shown). This suggests that the involved internal Ca²⁺ channels are not Ca²⁺-gated ryanodine receptors but either inositol trisphosphate receptors, which have also been observed in hippocampal neurons (Seymour-Laurent and Barish, 1995), or a different type of caffeine- and ryanodine-insensitive Ca²⁺ channel. The involvement of inositol trisphosphate receptors, however, is not likely, because they can be blocked by caffeine, at least in Xenopus oocytes (Parker and Ivorra, 1991).

Rapid kinetics and spatial analysis of intracellular Ca²⁺ amplification

We determined the time course of internal Ca²⁺ release and the location of release using a confocal microscope in line scan analysis mode (60 µsec scan time). As shown in Figure 3, the increase in Ca²⁺ concentration occurs over a period of 10-15 msec, much longer than the duration of an action potential (0.6-2.5 msec in hippocampal neurons) (Kandel et al., 1961; Peacock, 1979; Segal, 1983; Wiener et al., 1989; Bekkers and Stevens, 1991; Wheeler et al., 1996). The marked disparity between the time course of the Ca²⁺ rise and the duration of an action potential further strengthens the argument that most of the Ca²⁺ rise comes from release from internal stores and not from influx via voltage-gated Ca²⁺ channels. The time scale of 10-15 msec is similar to that observed for amplification mechanisms in smooth muscle, cardiac muscle, and skeletal muscle (Cannell et al., 1995; Nelson et al., 1995; Klein et al., 1996).

Figure 3A shows a spatial analysis of the Ca²⁺ release sites in a surface plot of the fluorescence intensity (z-axis) along a line across the soma (x-axis) as a function of time after the field pulse (y-axis). The [Ca²⁺]_i increases disproportionately more in regions close to the plasma membrane, suggesting that most of the Ca²⁺ release occurs in a region within <3 µm from the plasma membrane. Figure 3B shows such increases in fluorescence intensity in the region near the plasma membrane caused by three individual action potentials. Figure 3C shows a similar plot for an average of 16 action potentials (from nine neurons). The solid line is the best fit to the data, and the dashed line is the first derivative of the calculated curve fit. The highest rate of release occurred 3-5 msec after the onset of the field pulse, and Ca²⁺ release lasted for 10-15 msec. Delayed binding of Ca²⁺ to Fluo3 does not prolong the increase in fluorescence intensity significantly, because the K_on for Ca²⁺ binding by Fluo3 has been found to be 10⁹ M/sec or higher (Eberhard and Erne, 1989), suggesting that binding to the indicator occurs in <20 µsec (K_on × [Fluo3] with [Fluo3] assumed to be 50 µM).

Dependence of Ca²⁺ amplification on the number and frequency of action potentials

The properties of these internal Ca²⁺ release sites were investigated further by analyzing whether multiple action potentials cause potentiation or suppression of subsequent responses. Such a frequency dependence of Ca²⁺ responses may serve as a means for decoding electrical inputs within the soma. Physiologically meaningful repetition rates that have been observed in the hippocampus in vivo are in the range of 5-40 action potentials at frequencies of up to 50 Hz (Wiener et al., 1989). Interestingly, within the physiologically significant range, the peak [Ca²⁺]_i was strikingly linear with the number of action potentials (Fig. 4A). Figure 4B shows that the intracellular [Ca²⁺] response has a small dependence on the frequency of the field pulses, with a maximal [Ca²⁺] response triggered at ~40 Hz. The experiment in Figure 4B used 20 field pulses per burst to keep total stimulation time at 10 Hz to 2 sec and thereby to minimize any reduction in peak amplitude caused by the action of Ca²⁺ pumps.

---

Fig. 4. Dependence of internal Ca²⁺ release on the number and frequency of action potentials. This analysis shows that neither potentiation nor suppression of Ca²⁺ responses occurs over physiologically meaningful repetition rates (40 action potentials, <50 Hz). A, Linear dependence between the number of action potentials and the calculated peak intracellular free [Ca²⁺]. B, Intracellular [Ca²⁺] responses are maximal for frequencies of ~40 Hz. Peak Ca²⁺ responses after 20 field pulses are shown.
[View Larger Version of this Image (21K GIF file)]

---

Although the total amount of Ca²⁺ in the store was sufficient to cause a significant rise of [Ca²⁺] in the soma for a train of action potentials, each individual action potential caused an average somatic [Ca²⁺]_i to increase by 10 nM. Nevertheless, Figure 3A demonstrates that a local [Ca²⁺]_i close to the plasma membrane can rise significantly higher during a Ca²⁺ transient. Taken together, these observations suggest that internal Ca²⁺ release functions mostly as a linear amplification device with a small potentiation of the [Ca²⁺] response for frequencies of ~40 Hz.

---

DISCUSSION

Our data suggest that action potential-induced influx of Ca²⁺ into the soma results in a more than 30-fold amplification of the Ca²⁺ response by a 10- to 15-msec-long Ca²⁺ release process that requires Ca²⁺ stores close to the plasma membrane. This estimate is based on the observation that action potentials result in Ca²⁺ transients of identical amplitude in extracellular Ca²⁺ concentrations of 50 µM and 1.5 mM. This estimate also relies on the observed linear dependence of Ca²⁺ flux through voltage-gated Ca²⁺ channels as a function of the extracellular Ca²⁺ concentration (Frankenhaeuser and Hodgkin, 1957; Okamoto et al., 1976; Kawa, 1979; Hagiwara and Byerly, 1981). A second line of evidence suggesting a similarly large amplification process is indicated by the discrepancy between the concentration of La³⁺ necessary to block action potential-induced Ca²⁺ transients versus that needed to block individual voltage-gated Ca²⁺ channels. An amplification mechanism might be necessary, because Ca²⁺ is buffered between 100-fold in gonadotrophs (Tse et al., 1994) and 500- to 1000-fold in neurons (Muller et al., 1993; Llano et al., 1994), suggesting that a large influx or large internal release of Ca²⁺ ions is required to cause a substantial Ca²⁺ rise within the soma. Because adjacent neurons and glia are closely juxtaposed, and extracellular Ca²⁺ is buffered only weakly, there may be an insufficient number of extracellular Ca²⁺ ions available to cause a significant rise in [Ca²⁺]_i by Ca²⁺ influx alone. Therefore, the internal Ca²⁺ amplification mechanism identified in this study may play an important role in the production of significant Ca²⁺ signals in the neuronal soma.

In addition to the extensive buffering of Ca²⁺ in the cytosol and the small number of available extracellular Ca²⁺ ions, the Ca²⁺ responses are also limited by the volume-to-surface ratio, which is much larger in the soma than at the synapse, and would increase significantly the relative density of voltage-gated Ca²⁺ channels that would be required in the soma to generate a sizable Ca²⁺ response. Similar volume-to-surface limitations also exist for cardiac and skeletal muscle, in which intracellular Ca²⁺ channels are opened by Ca²⁺-induced ryanodine receptor opening (Cannell et al., 1995) and by an electromechanical coupling between the dihydropyridine receptor and the ryanodine receptor (Klein et al., 1996). Thus, internal somatic Ca²⁺ amplification can be used to amplify a short action potential-induced Ca²⁺ influx at the plasma membrane into a functionally significant Ca²⁺ response without requiring a large density of voltage-gated Ca²⁺ channels in the soma.

The induction of long-term neuronal plasticity is thought to rely on the control of transcriptional activity and often involves growth factors and other receptor ligands; however, neurons also need a means to regulate gene expression directly as a function of their electrical activity (Ghosh et al., 1994; Deisseroth et al., 1996). Somatic Ca²⁺ increases are likely to be important for this type of regulation. Ca²⁺-mediated changes in gene expression occur for Ca²⁺ increases in the cell body in the concentration range of a few hundred nanomolar to 1 µM, suggesting that the observed Ca²⁺ increases of 10 nM/action potential become physiologically meaningful if more than ~20 action potentials are triggered within 3 sec. Three seconds constitutes the time required for Ca²⁺ pumps to revert Ca²⁺ increases (see Fig. 1A). Comparable numbers of action potentials during such a period have been observed in in vivo recordings from hippocampal neurons (Wiener et al., 1989), suggesting that the Ca²⁺ amplification mechanism reported here can provide sufficiently large Ca²⁺ responses in physiologically relevant situations.

Another potential advantage of using intracellular Ca²⁺ amplification is that the cellular response to electrical inputs can be controlled better. Many functions of Ca²⁺ are thought to be localized by having effector proteins in the direct vicinity of Ca²⁺ channels (Schweizer et al., 1995). Furthermore, the gating of the known intracellular Ca²⁺ release channels is highly regulated by phosphorylation and other signaling events. Thus, by relying on intracellular Ca²⁺ release, the functional response to electrical inputs can be readily shaped by altering the intracellular localization of Ca²⁺ effectors and regulating the amount of internally released Ca²⁺.

---

FOOTNOTES

Received Jan. 31, 1997; revised March 4, 1997; accepted March 5, 1997.

  

This work was supported by National Institutes of Health Grants GM-48113 and GM-51457. J.M.J. was a Howard Hughes Medical Institute Medical Student Research Training Fellow. T.M. was supported by a fellowship from the David and Lucile Packard Foundation. We are grateful to Dr. C. Martenson for his work on the neuron cultures and for helpful advice and assistance. Dr. T. Ryan (Stanford University) was instrumental in setting up the neuronal culture. We also acknowledge J. Horne, E. Oancea, and Drs. M. Teruel, F. Schweizer, and J. Williams for support with the experiments and stimulating discussions.

Correspondence should be addressed to Dr. Tobias Meyer, Department of Cell Biology, Nanaline Duke Building, Room 346, Box 3709, Duke University Medical Center, Durham, NC 27710. 

---

REFERENCES

• Augustine GJ, Neher E (1992) Neuronal Ca²⁺ signalling takes the local route. Curr Opin Neurobiol 2:302-307[Medline].
• Basarsky TA, Parpura V, Haydon PG (1994) Hippocampal synaptogenesis in cell culture: developmental time course of synapse formation, calcium influx, and synaptic protein distribution. J Neurosci 14:6402-6411[Abstract].
• Bekkers JM, Stevens CF (1991) Excitatory and inhibitory autaptic currents in isolated hippocampal neurons maintained in cell culture. Proc Natl Acad Sci USA 88:7834-7838[Abstract/Free Full Text].
• Boland LM, Brown TA, Dingledine R (1991) Gadolinium block of calcium channels: influence of bicarbonate. Brain Res 563:142-150[ISI][Medline].
• Cannell MB, Cheng H, Lederer WJ (1995) The control of calcium release in heart muscle. Science 268:1045-1049[Abstract/Free Full Text].
• Deisseroth K, Bito H, Tsien RW (1996) Signaling from synapse to nucleus: postsynaptic CREB phosphorylation during multiple forms of hippocampal synaptic plasticity. Neuron 16:89-101[ISI][Medline].
• Eberhard M, Erne P (1989) Kinetics of calcium binding to Fluo-3 determined by stopped-flow fluorescence. Biochem Biophys Res Commun 163:309-314[ISI][Medline].
• Frank DA, Greenberg ME (1994) CREB: a mediator of long-term memory from mollusks to mammals. Cell 79:5-8[ISI][Medline].
• Frankenhaeuser B, Hodgkin AL (1957) The action of calcium on the electrical properties of squid axons. J Physiol 137:218-244.
• Ghosh A, Ginty DD, Bading H, Greenberg ME (1994) Calcium regulation of gene expression in neuronal cells. J Neurobiol 25:294-303[ISI][Medline].
• Hagiwara S, Byerly L (1981) Calcium channel. Annu Rev Neurosci 4:69-125[ISI][Medline].
• Kandel ER, Spencer WA, Brinley Jr FJ (1961) Electrophysiology of hippocampal neurons: sequential invasion and synaptic organization. J Neurophysiol 24:225-242[Free Full Text].
• Kawa K (1979) Zinc-dependent action potentials in giant neurons of the snail, Euhadra quaestia. J Membr Biol 49:325-344[ISI][Medline].
• Klein MG, Cheng H, Santana LF, Jiang YH, Lederer WJ, Schneider MF (1996) Two mechanisms of quantized calcium release in skeletal muscle. Nature 379:455-458[Medline].
• Llano I, DiPolo R, Marty A (1994) Calcium-induced calcium release in cerebellar Purkinje cells. Neuron 12:663-673[ISI][Medline].
• Malenka RC (1994) Synaptic plasticity in the hippocampus: LTP and LTD. Cell 78:535-538[ISI][Medline].
• Malgaroli A (1994) LTP expression: hanging like a yo-yo? Semin Cell Biol 5:231-241[Medline].
• Muller TH, Partridge LD, Swandulla D (1993) Calcium buffering in bursting Helix pacemaker neurons. Pflügers Arch 425:499-505[ISI][Medline].
• Neher E (1993) Spatial and temporal aspects of Ca changes in secretion control. Biochem Soc Trans 21:420-423[ISI][Medline].
• Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, Lederer WJ (1995) Relaxation of arterial smooth muscle by calcium sparks. Science 270:633-637[Abstract/Free Full Text].
• Okamoto H, Takahashi K, Yoshii M (1976) Two components of the calcium current in the egg cell membrane of the tunicate. J Physiol (Lond) 255:527-561[Abstract/Free Full Text].
• Parker I, Ivorra I (1991) Caffeine inhibits inositol trisphosphate-mediated liberation of intracellular calcium in Xenopus oocytes. J Physiol (Lond) 433:229-240[Abstract/Free Full Text].
• Peacock JH (1979) Electrophysiology of dissociated hippocampal cultures from fetal mice. Brain Res 169:247-260[ISI][Medline].
• Ryan TA, Smith SJ (1995) Vessicle pool mobilization during action potential firing at hippocampal synapses. Neuron 14:983-989[ISI][Medline].
• Schweizer FE, Betz H, Augustine GJ (1995) From vesicle docking to endocytosis: intermediate reactions of exocytosis. Neuron 14:689-696[ISI][Medline].
• Segal M (1983) Rat hippocampal neurons in culture: responses to electrical and chemical stimuli. J Neurophysiol 50:1249-1264[Abstract/Free Full Text].
• Seymour-Laurent KJ, Barish ME (1995) Inositol 1,4,5-triphosphate and ryanodine receptor distributions and patterns of acetylcholine- and caffeine-induced calcium release in cultured mouse hippocampal neurons. J Neurosci 15:2592-2608[Abstract].
• Tse A, Tse FW, Hille B (1994) Calcium homeostasis in identified rat gonadotrophs. J Physiol (Lond) 477:511-525[ISI][Medline].
• Wheeler DB, Randall A, Tsien RW (1996) Changes in action potential duration alter reliance of excitatory synaptic transmission on multiple types of Ca²⁺ channels in rat hippocampus. J Neurosci 16:2226-2237[Abstract/Free Full Text].
• Wiener SI, Paul CA, Eichenbaum H (1989) Spatial and behavioral correlates of hippocampal neuronal activity. J Neurosci 9:2737-2763[Abstract].

---

Copyright ©1997 Society for Neuroscience   0270-6474/1997/174129-07$05.00/0

This article has been cited by other articles:

				L. Chen, J. D. Bohanick, M. Nishihara, J. K. Seamans, and C. R. Yang Dopamine D1/5 Receptor-Mediated Long-Term Potentiation of Intrinsic Excitability in Rat Prefrontal Cortical Neurons: Ca2+-Dependent Intracellular Signaling J Neurophysiol, March 1, 2007; 97(3): 2448 - 2464. [Abstract] [Full Text] [PDF]

				V. Scheuss, R. Yasuda, A. Sobczyk, and K. Svoboda Nonlinear [Ca2+] Signaling in Dendrites and Spines Caused by Activity-Dependent Depression of Ca2+ Extrusion J. Neurosci., August 2, 2006; 26(31): 8183 - 8194. [Abstract] [Full Text] [PDF]

				D. Kiryushko, V. Novitskaya, V. Soroka, J. Klingelhofer, E. Lukanidin, V. Berezin, and E. Bock Molecular Mechanisms of Ca2+ Signaling in Neurons Induced by the S100A4 Protein. Mol. Cell. Biol., May 1, 2006; 26(9): 3625 - 3638. [Abstract] [Full Text] [PDF]

				J. C. Gant, M. M. Sama, P. W. Landfield, and O. Thibault Early and simultaneous emergence of multiple hippocampal biomarkers of aging is mediated by Ca2+-induced Ca2+ release. J. Neurosci., March 29, 2006; 26(13): 3482 - 3490. [Abstract] [Full Text] [PDF]

				M. L. Craske, M. Fivaz, N. N. Batada, and T. Meyer Spines and neurite branches function as geometric attractors that enhance protein kinase C action J. Cell Biol., September 26, 2005; 170(7): 1147 - 1158. [Abstract] [Full Text] [PDF]

				A. Verkhratsky Physiology and Pathophysiology of the Calcium Store in the Endoplasmic Reticulum of Neurons Physiol Rev, January 1, 2005; 85(1): 201 - 279. [Abstract] [Full Text] [PDF]

				A. Kumar and T. C. Foster 17beta -Estradiol Benzoate Decreases the AHP Amplitude in CA1 Pyramidal Neurons J Neurophysiol, August 1, 2002; 88(2): 621 - 626. [Abstract] [Full Text] [PDF]

				J. Meldolesi Rapidly Exchanging Ca2+ Stores: Ubiquitous Partners of Surface Channels in Neurons Physiology, August 1, 2002; 17(4): 144 - 149. [Abstract] [Full Text] [PDF]

				A. Gartner and V. Staiger Neurotrophin secretion from hippocampal neurons evoked by long-term-potentiation-inducing electrical stimulation patterns PNAS, April 30, 2002; 99(9): 6386 - 6391. [Abstract] [Full Text] [PDF]

				P. Mitra and M. M. Slaughter Mechanism of Generation of Spontaneous Miniature Outward Currents (SMOCs) in Retinal Amacrine Cells J. Gen. Physiol., April 2, 2002; 119(4): 355 - 372. [Abstract] [Full Text] [PDF]

				A. Aguilo, T. H. Schwartz, V. S. Kumar, Z. A. Peterlin, A. Tsiola, E. Soriano, and R. Yuste Involvement of Cajal-Retzius Neurons in Spontaneous Correlated Activity of Embryonic and Postnatal Layer 1 from Wild-Type and Reeler Mice J. Neurosci., December 15, 1999; 19(24): 10856 - 10868. [Abstract] [Full Text] [PDF]

				M. Tomic, T.-a. Koshimizu, D. Yuan, S. A. Andric, D. Zivadinovic, and S. S. Stojilkovic Characterization of a Plasma Membrane Calcium Oscillator in Rat Pituitary Somatotrophs J. Biol. Chem., December 10, 1999; 274(50): 35693 - 35702. [Abstract] [Full Text] [PDF]

				N. Volfovsky, H. Parnas, M. Segal, and E. Korkotian Geometry of Dendritic Spines Affects Calcium Dynamics in Hippocampal Neurons: Theory and Experiments J Neurophysiol, July 1, 1999; 82(1): 450 - 462. [Abstract] [Full Text] [PDF]

				D. J. Fickbohm and A. L. Willard Upregulation of Calcium Homeostatic Mechanisms in Chronically Depolarized Rat Myenteric Neurons J Neurophysiol, June 1, 1999; 81(6): 2683 - 2695. [Abstract] [Full Text] [PDF]

				V. M. Sandler and J.-G. Barbara Calcium-Induced Calcium Release Contributes to Action Potential-Evoked Calcium Transients in Hippocampal CA1 Pyramidal Neurons J. Neurosci., June 1, 1999; 19(11): 4325 - 4336. [Abstract] [Full Text] [PDF]

				J. Meldolesi and T. Pozzan The Heterogeneity of ER Ca2+ Stores Has a Key Role in Nonmuscle Cell Signaling and Function J. Cell Biol., September 21, 1998; 142(6): 1395 - 1398. [Full Text] [PDF]

				A. M. Horgan and P. F. Copenhaver G Protein-Mediated Inhibition of Neuronal Migration Requires Calcium Influx J. Neurosci., June 1, 1998; 18(11): 4189 - 4200. [Abstract] [Full Text] [PDF]

				P. Mitra and M. M. Slaughter Mechanism of Generation of Spontaneous Miniature Outward Currents (SMOCs) in Retinal Amacrine Cells J. Gen. Physiol., April 2, 2002; 119(4): 355 - 372. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (34) Citing Articles via Google Scholar Google Scholar Articles by Jacobs, J. M. Articles by Meyer, T.